The adrenocorticotropic hormone (ACTH) receptor, also known as the melanocortin-2 receptor (MC2R), is a subtype of the melanocortin receptor family, which plays an important role in regulating and maintaining adrenocortical function, specifically steroidogenesis. Mutations of the human MC2R (hMC2R) gene have been demonstrated in human with familial glucocorticoid deficiency. It presents in childhood with failure-to thrive, weakness, fatigue and possible adrenal crisis. Therefore, a better understanding of the molecular basis of hMG2R is important to understanding the physiology and pathology of hMC2R function. It is also critical in developing effective therapeutic strategies for the treatment of important human adrenal disorders. The hMC2R is homologous to the other 4 cloned melanocortin receptors, which together form a distinct subfamily of 7 transmembrane domain G protein-coupled receptors (GPCR). Within the past several years we and other investigators have performed extensive studies to determine the molecular basis of ligand receptor interaction between the melanocortin family of peptides (including agonist a-MSH, ACTH, and antagonist Agouti signaling protein (ASIP), Agouti-related protein (AGRP)) and the melanocortin receptors. However, the molecular determinants of MC2R responsible for agonist ACTH and antagonist ASIP binding and action remain unclear. This proposal is directed towards examining the molecular determinants of hMC2R responsible for ligand binding and receptor activation. Based on our previous work with other melanocortin receptors, we hypothesize that: 1) transmembrane domains of hMC2R play important roles in agonist ACTH binding and receptor activation and 2) both extracellular loops and transmembrane domains of hMC2R are crucial for antagonist ASIP binding and activity. To test this hypothesis we will utilize the hMC2R mutagenesis and chimeric receptor studies to determine the molecular basis of hMC2R ligand receptor interaction. The proposed studies will provide important information needed to fill in the gap in our current knowledge of the molecular determinants of hMC2R responsible for ligand binding and receptor activation. The information will serve to establish a foundation on which to build a comprehensive understanding of the signaling events that regulate ACTH mediated adrenal function in physiologic and diseased states.